Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.

The glutathione S-transferases (GST) represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST), whereas the membrane-bound enzymes, microsomal GST and leukotriene C, synthetase, are encoded by single genes and both have arisen separately from the soluble GST. Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals. In this article the biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress.A description of the mechanisms of transcriptional and posttranscriptional regulat...

The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.

In this review, we have examined the biochemical and toxic responses produced by halogenated aromatic hydrocarbons and have tried to develop a model for their mechanism of action. These compounds bind to a cellular receptor and evoke a sustained pleiotropic response. In many tissues this response consists of the expression of a battery of enzymes which are, for the most part, involved in drug metabolism, but in other tissues, those which develop toxicity, an additional set of genes is expressed which effects cellular involution, division, and/or differentiation. The toxicity of these compounds appears to be due to the sustained expression of a normal cellular regulatory system, of which we were previously unaware. In future investigations it is hoped that we will learn the nature and physiologic role of this regulatory system. Only then can we hope to understand the mechanism of toxicity of these compounds.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin and Related Halogenated Aromatic Hydrocarbons: Examination of the Mechanism of Toxicity

Publisher Summary This chapter provides the spectrophotometric, titrimetric, nitrite, and cyanide assay for the differentiation of glutathione S-transferases. Spectrophotometric assays depend upon a direct change in the absorbance of the substrate when it is conjugated with glutathione (GSH). Because each of the reactions is catalyzed at a finite rate in the absence of enzyme, care is needed to reduce nonenzymatic catalysis by minimizing substrate concentrations and by decreasing pH wherever necessary. Titrimetric assay is based on the principle that the conjugation of alkyl halides with GSH can be measured titrimetrically. Although acid production accompanies many of the transferase catalyzed reactions in which thioethers are formed, titrimetry is only used when more convenient assays are not available. Nitrite assay is based on the principle that nitrite is released when GSH reacts with nitroalkanes or with organic nitrate esters. The nitrite can be assayed as the limiting factor in a diazotization reaction with sulfanilamide that produces a readily quantitatable pink dye. Cyanide assay is based on the fact that when glutathione transferases catalyze the attack of the glutathione thiolate ion on the electrophilic sulfur atom of several organic thiocyanates, it results in the formation of an asymmetric glutathionyl disulfide and cyanide. Cyanide can be readily quantitated by a calorimetric method.

Assays for differentiation of glutathione S-transferases.

Glutathione metabolism and its selective modification.

Molecular basis for the lack of bilirubin-specific and 3-methylcholanthrene-inducible UDP-glucuronosyltransferase activities in Gunn rats. The two isoforms are encoded by distinct mRNA species that share an identical single base deletion.

Transport of reduced glutathione (GSH) and its derivatives was studied in rat-liver sinusoidal plasma-membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited transport of GSH into an osmotically active intravesicular space. Equilibrium uptake of vesicle-associated GSH was similar to that of free ligands which can be trapped by the intravesicular space of sinusoidal membrane samples. Kinetic analysis of the transport process revealed that the sinusoidal membrane vesicles have high-affinity and low-affinity GSH transport systems ; the former has an apparent K, of 0.34 mM for GSH and V of 1.4 nmol x mg protein-' x 20 s-', and the latter has an apparent X, of 3.3 mM and Vof 3.9 nmol x mg protein-' x 20 s-'. Both K, values are lower than intrahepatic GSH levels, indicating that GSH transport across the sinusoidal membrane occurs via carrier-mediated mechanism and argues simple diffusion mechanism. The presence of oxidized glutathione (GSSG) or S-benzylglutathione inhibited GSH transport by the vesicles; the low-affinity transport system was inhibited more markedly than the high-affinity transport system. This suggests that these glutathione derivatives interacted preferentially with the low-affinity transport system for GSH. S-Dinitrophenylglutathione was also transported by the vesicles by a process which was inhibited by GSH and GSSG. The vesicles also transported GSSG, and this transport was markedly inhibited by S-benzylglutathione or GSH. The transport systems in sinusoidal plasma membranes may function in vivo in translocating GSH and its derivatives from hepatocytes into plasma and play an important role in inter-organ metabolism of these compounds. GSH occurs intracellularly with high concentrations (z 10 mM) [l]. Hepatic GSH is translocated from hepatocyte into bile and plasma [2]. In isolated perfused liver, 14 nmol of hepatic GSH x min- ' x g liver-' appeared in the effluent perfusate [3]. The plasma GSH level is low (z27 pM) [4] presumably due to rapid utilization by tissues which have y-glutamyltransferase [5, 61. Kidney has the highest y-glutamyltransferase activity [7] and is the major organ for extraction of plasma GSH [S]. Schulman et al. [S] described a patient with y-glutamyltransferase deficiency who had glutathionemia and glutathionuria; about 0.85 g of glutathione was excreted per day in urine of this patient. Thus, translocation of GSH from hepatocytes and probably from other cells is important in cellular turnover of GSH. Glutathione S-conjugates of xenobiotics and GSSG are secreted preferentially into bile. However, these derivatives also come out of the liver into effluent perfusate when their intracellular levels increased [9]. The mechanism of hepatic transport of GSH and its derivatives across the hepatocyte sinusoidal plasma membranes is not known. Solute transport across plasma membranes of intact cells is a complex system in which transmembrane process are affected by intracellular and extracellular metabolic conditions. Isolated plasma membrane vesicles provide a simple system in which membranous process can be studied independently from cellular metabolic events. Previous studies [lo] reported isolation of rat liver sinusoidal plasma membrane vesicles which retain Na+-cotransport systems for bile acids and alanine.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1984.tb07943.x

Glutathione transport across hepatocyte plasma membranes. Analysis using isolated rat-liver sinusoidal-membrane vesicles.

Ligandin. Bilirubin binding and glutathione-S-transferase activity are independent processes.

Pregnancy, Oral Contraceptives, and Chronic Familial Jaundice with Predominantly Conjugated Hyperbilirubinemia (Dubin-Johnson Syndrome)

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine–labeled GSH into an osmotically reactive intravesicular space. Althought the canalicular membrane vesicles possess γ-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle associated transferase by affinity labeling with L-(αS,5,S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07590.x

Irwin M. Arias

Papers

Molecular basis for the lack of bilirubin-specific and 3-methylcholanthrene-inducible UDP-glucuronosyltransferase activities in Gunn rats. The two isoforms are encoded by distinct mRNA species that share an identical single base deletion.

Glutathione transport across hepatocyte plasma membranes. Analysis using isolated rat-liver sinusoidal-membrane vesicles.

Ligandin. Bilirubin binding and glutathione-S-transferase activity are independent processes.

Pregnancy, Oral Contraceptives, and Chronic Familial Jaundice with Predominantly Conjugated Hyperbilirubinemia (Dubin-Johnson Syndrome)

The Mechanism of Biliary Secretion of Reduced Glutathione